Directed Evolution - A Typical Experiment

A Typical Experiment

A typical directed evolution experiment involves three steps:

1. Diversification: The gene encoding the protein of interest is mutated and/or recombined at random to create a large library of gene variants. Techniques commonly used in this step are error-prone PCR and DNA shuffling.
2. Selection: The library is tested for the presence of mutants (variants) possessing the desired property using a screen or selection. Screens enable the researcher to identify and isolate high-performing mutants by hand, while selections automatically eliminate all nonfunctional mutants.
3. Amplification: The variants identified in the selection or screen are replicated manyfold, enabling researchers to sequence their DNA in order to understand what mutations have occurred.

Together, these three steps are termed a "round" of directed evolution. Most experiments involve more than one round. In these experiments, the "winners" of the previous round are diversified in the next round to create a new library. At the end of the experiment, all evolved protein or RNA mutants are characterized using biochemical methods.